Oxidative stress is defined as an imbalance between pro-oxidants
and anti-oxidants, resulting in damage to cell by reactive oxygen
species (ROS). Reactive oxygen species include oxygen ions, free
radicals and peroxides. They form as a natural byproduct of the
normal metabolism of oxygen and have important roles in a number
of biological processes, such as the killing of bacteria. During
times of environmental stress ROS levels can increase dramatically
which can result in significant damage to cell structures, especially
in absence of anti-oxidant defences, such as the enzymes superoxide
dismutase, catalase, glutathione peroxidase and glutathione reductase
or antioxidant vitamins A, C and E and polyphenol antioxidants.
Vitamin E plays an important role in cellular defence against
lipid peroxidation – a degradation of cell membrane by free
radicals.

There is mounting evidence that abnormalities of ROS and nitric
oxide (NO) may underlie a wide range of neuropsychiatric disorders.
Abnormal methionine metabolism, high levels of homocysteine and
oxidative stress are also generally associated with neuropsychiatric
disorders. NO signalling has been implicated in a number of physiological
functions such as noradrenaline and dopamine releases. It is thought
to have neuroprotective effects at low to moderate concentrations,
but excessive NO production can cause oxidative stress to neurons
thus impairing their function.

Studies comparing the level of homocysteine and
other biomarkers in children with autism to controls showed that
in children with autism there were highter levels of homocysteine,
which was negatively correlated with glutathione peroxidase activity,
low human paraoxonase 1 arylesterase activity, suboptimal levels
of vitamin B 12 [16297937,
12445495]
and increased levels of NO [12691871,
14960298].

Lipid peroxidation was found to be elevated in
autism indicating increased oxidative stress. Moderate to dramatic
increases in isoprostane levels [16081262,
16908745],
decreased levels of phosphatidylethanolamine and increased levels
phosphatidylserine [16766163]
were observed in children with autism as compared to controls.
Levels of major antioxidant proteins transferrin
(iron-binding protein) and ceruloplasmin (copper-binding
protein) were found to be significantly reduced in sera of autistic
children. A strong correlation was observed between reduced levels
of these proteins and loss of previously acquired language skills
[15363659].

Another study measured levels of metabolites in methionine pathways
in autistic children and found that plasma methionine and the
ratio of S-adenosylmethionine (SAM) to S-adenosyl-homocysteine
(SAH), an indicator of methylation capacity,
were significantly decreased in the autistic children relative
to controls. In addition, plasma levels of cysteine, glutathione,
and the ratio of reduced to oxidized glutathione,
indicative of antioxidant capacity and redox homeostasis, were
significantly decreased in autistic group. The same study evaluated
common polymorphic variants known to modulate these metabolic
pathways in 360 autistic children and 205 controls. Differences
in allele frequency and/or significant gene-gene interactions
were found for relevant genes encoding the reduced folate carrier
(RFC 80G), transcobalamin II (TCN2 776G), catechol- O-methyltransferase
(COMT 472G), methylenetetrahydrofolate reductase (MTHFR 677C and
1298A), and glutathione-S-transferase (GST M1). The authors propose
that an increased vulnerability to oxidative stress
may be a contributive factor to the development and clinical manifestations
of autism [16917939].

Oxidative
damage in autism is also associated with altered expression of
brain neurotrophins critical for normal brain growth and differentiation.
An increase in 3-nitrotyrosine (3-NT), a marker of oxidative stress
damage to proteins in autistic cerebella has been reported. Altered
levels of brain NT-3 are likely to contribute to autistic pathology
not only by affecting brain axonal targeting and synapse formation
but also by further exacerbating oxidative stress and possibly
contributing to Purkinje cell abnormalities (19357934).

A study looking into cellular and mitochondrial glutathione redox
imbalance in lymphoblastoid cells derived from children with autism
found that, compared to controls, autism LCLs exhibit a reduced
glutathione reserve capacity in both cytosol and mitochondria
that may compromise antioxidant defense and detoxification capacity
under prooxidant conditions (19307255).

Several murine studies showed that defective homocysteine remethylation
can be caused by deficiency of either methionine synthase enzyme
that catalyzes the folate-dependent remethylation of homocysteine
to methionine or deficiency of folic acid that produces oxidative
stress and endothelial dysfunction in the cerebral microcirculation.
[16043641].

In addition to genetic defects such as cystathionine beta-synthase
(CBS) or MTHFR, additional factor that can contribute to increasing
plasma homocysteine levels is the nutritional status of vitamin
B12, vitamin B6, or folate deficiencies.

Folate deficiency induces neurotoxicity by multiple
routes – apart from increasing oxidative stress by increasing
the levels of homocysteine, it can also contribute to increases
in cytosolic calcium and to subsequent mitochondrial and DNA damage
(see Mitochondria). Folate deprivation was shown to induce calcium
influx initially through the LTCC, and subsequently through NMDA
channels and from internal stores [15038821].

Oxidative stress and calcium signalling

Recent data supports the speculation that calcium and reactive
oxygen species are two cross-talking messengers in various cellular
processe. The results of various studies have shown that calcium
is essential for production of ROS. Elevation of intracellular
calcium level is responsible for activation of ROS-generating
enzymes and formation of free radicals by the mitochondria
respiratory chain. It has been shown that the effective macrophage
redox defense against Chlamydia pneumoniae depends on LTCC channel
activation [12736823].
Hydrogen peroxide, a membrane-permeable form of reactive oxygen
species, was shown to enhance invard calcium current in cultured
dentate granule cells. This enhancement was cancelled by glutathione,
an antioxidant, and nifedipine, an LTCC channel blocker, suggesting
that oxidative stress induced by hydrogen peroxide selectively
regulates the activity of LTCC [14746893,
9152045].
Nimodipine, another calcium channel blocker, was also shown to
suppress ROS formation [10601165,
15820440,
9489715],
and verapamil was both protective against oxidative stress and
ameliorated morphological changes and dysfunction of mitochondria
(see Mitochondria) [16644187].

On the other hand, an increase in intracellular calcium concentration
may be stimulated by ROS. Hydrogen peroxide has been recently
shown to accelerate the overall channel opening process in voltage-dependent
calcium channels in plant and animal cells. In addition to outer
membrane calcium channels, IP3 receptors as well as the ryanodine
receptors of sarcoplasmic reticulum have also been demonstrated
to be redox-regulated [14616077].

The rise in intracellular calcium activates,
amongst other things, nitric oxide synthetases,
a group of enzymes responsible for the synthesis of nitric oxide.
A study looking into mechanisms of NO synthase in the developing
rat cortex found that, quote: “… depolarization
following GABA-A receptor activation leads to opening of L-type
voltage-gated calcium channels, resulting in an increased calcium
influx, which in turn leads to phosphorylation and, thus, activation,
of the transcription factor CREB; the phosphorylated CREB can
then induce BDNF, as well as nNOS " [14604759,
9153595]
(see also Brain_Development).

In another study, application of dihydropyridine calcium channel
blockers had protective effects against endothelial cell oxidative
injury due to combined nitric oxide and superoxide. Nisoldipine,
nicardipine and nifedipine all attenuated oxidative-insult induced
by loss of reduced glutathione, with nisoldipine demonstrating
greatest protection [11820858].

It has been shown that in red blood cells increases in intracellular
calcium contentrations lead to a decrease of membrane protein
methyl esterification and a subsequent impairment of S-adenosylmethionine
synthesis (SAM). After the removal of extra calcium from the cells
the levels of methyl esterification returned to normal [3081340].

Excessive lipid peroxidation is implicated in the pathogenesis
of neurodegenerative disorders and is brought apon by free radicals
action on cell membrane in the absence of inadequate antioxidant
defence. Lipid peroxidation has been shown to modulate the activity
of VGCC. In one study a prolonged exposure to a lipid-peroxidation
enhancer resulted in neuronal death, which was prevented by treatment
with glutathione and attenuated by the LTCC blocker nimodipine.
It was concluded that the modulation of calcium channel activity
in response to lipid peroxidation may play important roles in
the responses of neurons to oxidative stress in both physiological
and pathological settings [12006588].

Homocysteine is found to overstimulate NMDA receptors, leading
to excessive calcium influx and possible neuronal damage [10797837].

Similar to the abovementioned cross talk between ROS and calcium,
glutathione, as well as being influenced by calcium,
also seems to have a critical role in gating the VGCC. Inhibition
of glutathione reductase by carmustine in vitro resulted in depletion
of glutathione and oxidative stress, and an influx of extracellular
calcium through LTCC. This increase in intracellular calcium was
dependent on the presence of extracellular calcium and could be
inhibited by calcium blockers nimodipine or nitrendipine. In addition,
this effect was also suppressed in cells that were treated with
an antioxidant deferoxamine, and enhanced in cells that were pretreated
with an inhibitor of glutathione synthesis, buthionine sulfoximine
[15321730].

It may be of relevance to note that calcium channels in pancratic
islets are very sensitive to levels of glutathione. Membrane thiols
are thought to play an important role in insulin sectretion due
to their effect on calcium influx via those channels [2424631].
(see Pancreatic function/GI)

Causes of oxidative stress

Apart from poor nutritional status and/or genetic factors, various
enviromental agents have also been implicated as causative agents
in disturbances in methylation pathways and increased oxidative
stress. Oxidative damage due to increased generation of reactive
oxygen species and reactive nitrogen species is a feature of many
viral infections. The increasing prevalence of
HIV-associated cognitive impairment has been the subject of many
recent studies, the result of which provide overwhelming evidence
for oxidative stress in mediating neuronal injury in patients
with HIV induced dementia. These studies also suggest that patients
with apolipoprotein E4 allele are more susceptible
to neuronal oxidative damage [17034352].

Raised homocysteine levels alongside folate deficiency has been
observed in HIV infected children [11737242]
and in other viral and mycoplasmal infections, including influenza
A and B, human parvovirus, rubella, infectious mononucleosis and
Mycoplasma pneumoniae [3033086]
[12214730].

One study looking at absorbtion of folate in HIV infected patients
has found that absorption of folic acid appears
to be significantly impaired in HIV disease, irrespective of the
stage of the disease or gastro-intestinal complaints. The authors
presented data to support their hypothesis that the virus can
cause an enteropathy in the absence of opportunist infection [1680150].

Hepatitis C induced oxidative stress is also widely studied, with
numerous studies showing Hepatitis infection causing a state of
chronic oxidative stress. These viruses have been associated with
changes in mitochondrial structure and function, including increased
calcim uptake [16958669].
(see Mitochondria)

Various enviromental toxins have been shown to
cause oxidative damage to the cell. Metal-mediated formation of
free radicals causes various modifications to DNA bases, enhanced
lipid peroxidation, and altered calcium and sulfhydryl
homeostasis. For a group of metals that include mercury, cadmium
and nickel, one route for their toxicity is depletion of glutathione
and bonding to sulfhydryl groups of proteins [15892631]
[8512585].

A study looking at the effects of both methylmercury and inorganic
mercury on cell oxidative stress and intracellular calcium concentration
in rat cerebellar granule neuron cultures tested neuroprotective
effects of several agents that selectively interfere with these
mechanisms. The results suggested that disruption of redox equilibrium
and calcium homeostasis contribute equally to inorganic mercury
cell damage, whereas oxidative stress is the main cause of methylmercury
neurotoxicity [11599010].

A study looking at functional activities of insulin-like growth
factor-1, dopamine-stimulated methionine synthase, and folate-dependent
methylation of phospholipids in normal development found that
these pathways were interrupted by neurodevelopmental toxins,
such as ethanol and heavy metals. Study results raised the possibility
that these toxins might exert adverse effects on methylation pathways
[14745455].
Reversible alterations of oxidative stress biomarkers resulting
from in utero and neonatal exposures of airborne manganese have
also been documented [16943606].
(see also Toxic_Agents)

Additonal issues for consideration:
implications of calcium signalling in oxalate formation and
deficiencies of magnesium and B vitamins

Deficiencies of several forms of vitamins B have been shown in
vitro and in rodent studies to enhance calcium influx through
VGCC. For example it has been observed that depolarization and
activation of LTCC occur during experimental thiamine deficiency,
and it was proposed that this mechanim may play a role in histological
brain lesions/damage induced by thiamine deficiency [9669323].

In the context of reports on beneficial effects of supplementiation
of vitamin B12 in autism, as well as simultaneous supplementation of
magnesium and vitamin B6 [links],
it is of interest that several studies observed that simultaneous
deficiencies of magnesium and pyridoxine may in fact act synergistically
on impairing the function of LTCC. Deficient rats were found to
exibit excessive influx of calcium into the intracellular compartments.
Lowering magnesium in resulted in elevation of calcium in cultured
canine celebral vascular cells, and this calcium entry could be
blocked by exposing the cells to vitamins B6, B12, or folic acid,
simultaneously or individually [10553943,
1645979, 9823019].

Of some relevance could be the observation that simulaneous application
of magnesium and pyridoxine significantly decreased formation
of oxalate in a small-scale human study [7992461].
It is proposed that regulation of calcium fluxes through LTCC
by these agents may at least in part underlie their effects in
mammals, as it does in plants [link],
as application of various calcium channel blockers has been shown
to decrease oxalate formation in human and in animal studies.
[8322624,
1845698,
10354288]

Conclusion:
Following the above findings it is suggested that folate and cobalamin
deficencies observed in neurological disorders may be caused by
a combination of genetic polymorphisms as well as impaired absorbtion
and disturbances in metabolic pathways due to viral and other
infections and enviromental toxic load, mediated in great part
through enhanced calcium signalling. The involvement of viral
infections and enviromental toxins as causes of disturbed folate
metabolism and oxidative stress and their relation to calcium
homeostasis may be of relevance to autism and neurological disorders
and requires further investigation.